Bioresource Technology 99 (2008) 8532–8536

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Short Communication

Enhanced hyaluronic acid production by a two-stage culture strategy based on

the modeling of batch and fed-batch cultivation of Streptococcus zooepidemicus
Long Liu a, Guocheng Du a,b,*, Jian Chen a,b,*, Miao Wang c, Jun Sun d
a
School of Biotechnology, Jiangnan University, Wuxi 214122, China
b
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China
c
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
d
Institute of Information Technology, Jiangnan University, Wuxi 214122, China

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to enhance hyaluronic acid (HA) production by a two-stage culture strategy based on
Received 2 January 2008 the modeling of batch and fed-batch culture of Streptococcus zooepidemicus. Batch culture had higher spe-
Received in revised form 24 February 2008 cific HA synthesis rate while fed-batch culture had higher specific cell growth rate. The lower specific HA
Accepted 27 February 2008
synthesis rate in fed-batch culture resulted from the competition of cell growth for the common precur-
Available online 7 April 2008
sors at a low substrate concentration. Based on the modeling of batch and fed-batch culture of S. zooep-
idemicus, a two-stage culture strategy was proposed to enhance HA production. S. zooepidemicus were
Keywords:
cultured in a fed-batch mode with sucrose concentration maintained at 1.0 ± 0.2 g/L during 0–8 h and
Modeling
Two-stage culture
then batch culture was performed during 8–20 h with an initial sucrose concentration of 15 g/L. With
Hyaluronic acid the proposed two-stage culture strategy, HA production was increased to 6.6 g/L compared with 5.0 g/
Streptococcus zooepidemicus L in batch culture with the same total sucrose. The enhanced HA production by the proposed two-stage
culture strategy resulted from the decreased inhibition of cell growth and the increased transformation
rate of sucrose to HA.
Ó 2008 Published by Elsevier Ltd.

1. Introduction
Hyaluronic acid (HA) is a linear glycosaminoglycan polysaccha-
ride composed of repeating disaccharide units of alternative D-glu-
curonic acid (GlcUA) and N-acetylglucosamine (GlcNAc) (Laurent
et al., 1996). With unique physico-chemical properties, such as bio-
compatibility, lubricity and hydrophilicity, HA has been widely ap-
plied in biomedical, food, healthcare and cosmetic fields (Esposito
et al., 2005; Lapcik et al., 1998; Morra, 2005; Park et al., 2003; Pey-
ron, 1993).
Conventionally HA was extracted from animal tissues like roos-
ter combs, and now is increasingly produced by fermentation of
Streptococcus zooepidemicus with the advantages of low production
cost. Batch culture is a usual method for HA production, and there
are many studies concerning the optimization of culture conditions
in batch culture (Johns et al., 1994; Kim et al., 1996, 2006; Krahulec
and Krahulcová, 2006). However, a major disadvantage of batch
culture is that the production of aimed metabolites is limited
* Corresponding authors. Address: School of Biotechnology, Jiangnan University,
Wuxi 214122, China. Tel./fax: +86 510 85918309 (G. Du); tel.: +86 510 85913661;
fax: +86 510 85910799 (J. Chen).
E-mail addresses: gcdu@jiangnan.edu.cn (G. Du), jchen@jiangnan.edu.cn
(J. Chen).
due to the strong inhibitions caused by the substrates of high con-
centration (Ding and Tan, 2006).
With the advantage of reducing the time spent on reactor turn-
over, continuous cultivation is attracting more and more attentions
to produce HA (Fong Chong et al., 2005). However, continuous cul-
ture is not a perfect choice for HA production due to the instability
of the HA-producing phenotype of highly encapsulated strepto-
cocci strains in high dilution rates (Armstrong et al., 1997).
Fed-batch culture is a batch culture fed continuously or sequen-
tially with substrate without the removal of fermentation broth.
Fed-batch culture is generally superior to batch and continuous
culture, and is especially beneficial when changing nutrient con-
centrations affects the productivity of the desired products (Son
et al., 2007). However, as far as we know, there are few system-
atic reports on the production of HA by fed-batch culture of S.
zooepidemicus. In addition, we have only found two reports about
the modeling of HA fermentation process in the literature. Cooney
et al. (1999) developed a structured, two-compartment model for
HA fermentation in anaerobic and aerated conditions. Huang
et al. (2007) developed an unstructured model for cell growth
and HA production in batch culture. The model developed by Coo-
ney et al. (1999) is obviously a complicated one and not suitable for
process control. The model developed by Huang et al. (2007)
mainly dealt with the relationship between cell growth and HA

0960-8524/$ - see front matter Ó 2008 Published by Elsevier Ltd.

doi:10.1016/j.biortech.2008.02.035
 L. Liu et al. / Bioresource Technology 99 (2008) 8532–8536
Nomenclature
l specific growth rate (h1)
lmax maximum specific growth rate (h1)
S substrate concentration (g/L)
KS substrate saturation constant (g/L)
KI substrate inhibition constant (g/L)
P product concentration (g/L)
KIP product inhibition constant (g/L)
PLA concentration of lactic acid (g/L)
PHA concentration of HA (g/L)
KILA inhibition constant of lactic acid on cell growth (g/L)
aHA coupling constant of HA synthesis with cell growth
aLA coupling constant of lactic acid with cell growth
KHA inhibition constant of lactic acid on HA synthesis (g/L)
KLA inhibition constant of lactic acid on the synthesis of
itself (g/L)
synthesis, while the inhibition of substrate and lactic acid on cell
growth and HA synthesis was not considered. Therefore, a simple
and comprehensive modeling of HA fermentation process is neces-
sary for the process control and optimization.
The present study aimed to enhance HA production by a two-
stage cultivation strategy based on the modeling of batch and
fed-batch culture of S. zooepidemicus. Firstly, the modeling of batch
culture of S. zooepidemicus was carried out. Then the influence of
fed-batch culture on HA production was studied and modeled. Fi-
nally, a two-stage culture strategy was proposed to enhance HA
production based on the modeling of batch and fed-batch culture
of S. zooepidemicus.
2. Methods
2.1. Microorganism and media
S. zooepidemicus WSH-24 was used in this study. Fresh slants
were cultured at 37 °C for 12 h and were used for inoculation.
Seed culture medium consisted of (g/L): sucrose 20, yeast
extract 20, MgSO47H2O 2.0, MnSO44H2O 0.1, KH2PO4 2.0, CaCO3
20 and 1 mL trace elements solution. The trace element solution
consisted of (g/L): CaCl2 2.0, ZnCl2 0.046 and CuSO45H2O 0.019.
Fermentation medium contained (g/L): yeast extract 25, sucrose
70, K2SO4 1.3, MgSO47H2O 2.0, Na2HPO412H2O 6.2, FeSO47H2O
0.005 and 2.5 mL trace element solution (pH 7.2). Culture medium
was sterilized at 121 °C for 15 min.
2.2. Batch and fed-batch culture of S. zooepidemicus in a 7-L
fermentor
HA production by batch culture of S. zooepidemicus was carried
out with an initial sucrose concentration of 70 g/L. One loop of cells
from a fresh slant was transferred to 50 mL seed culture medium
and cultured on a rotary shaker at 200 rpm and 37 °C for
12 h. The seed culture was inoculated into a 7-L fermentor (Model
KL-7L, K3T Ko Bio Tech, Korea) with a working volume of 4.0 L.
The pH was automatically controlled at 7.0 by adding 5 mol/L
NaOH solution and temperature was maintained at 37 °C. The
aeration rate and agitation speed were 0.5 vvm and 200 rpm,
respectively.
The fed-batch culture with constant feeding rate was carried
out and was initiated as a batch culture with an initial sucrose con-
centration of 10 g/L. After 2 h of batch culture, the feeding sucrose
(600 g/L) was pumped into the fermenter at a feeding rate of
8533
t culture time (h)
YX the yield of cell on sucrose (g cell/g sucrose)
YHA the yield of HA on sucrose (g HA/g sucrose)
YLA the yield of lactic acid on sucrose (g lactic acid/g su-
crose)
me maintenance constant (h1)
X cell concentration (g/L)
Sopt the optimal sucrose concentration for cell growth (g/L)
V broth volume (L)
F feeding rate (mL/h)
cHA specific HA synthesis rate of (g HA/g cell/h)
cLA specific lactic acid synthesis rate (lactic acid/g cell/h)
k specific sucrose consumption rate (g sucrose/g cell/h)
22.5 mL/h by a peristaltic pump coupled with a computer. The
feeding rate of 22.5 mL/h was selected to make the total sucrose
in batch and fed-batch culture to be equal. The initial and final
broth volumes for fed-batch culture were 3.2 and 4.0 L,
respectively.
In the two-stage culture strategy, by the feedback control of re-
dox potential, the sucrose concentration was maintained at Sopt un-
til the cell concentration reached the maximum cell concentration
of batch culture. Then the batch culture of S. zooepidemicus in the
second stage was started with an initial sucrose concentration of
15 g/L.
2.3. Analytical methods
Five milliliter of culture broth was mixed with two folds of alco-
hol, followed by centrifugation at 560g for 10 min. Supernatant
was collected for further analysis of lactic acid and residual su-
crose. Lactic acid concentration was determined by an Agilent
1100 high-pressure liquid chromatograph (Agilent Technologies
Incorporation, USA) equipped with an Agilent G1313A injector
and an Agilent G1314A detector at 215 nm. The supernatant was
filtered with 0.2 lm pore size filter prior to injection into a C18 col-
umn (4.6  200 mm). 0.5 mol/L KH2PO4 (pH 2.5) was used as mo-
bile phase at a flow rate of 1 mL/min. The temperature of the
column was maintained at 30 °C.
Sucrose concentration was determined using the resorcinol
method (Ning, 1998). Simply, 0.9 mL of sucrose sample was mixed
with 0.1 mL of NaOH solution (2 mol/L), and the solution was
heated for 10 min in boiling-water bath. Then 1 mL of resorcinol
solution and 3 mL of HCl (10 mol/L) were added into the cooled
sample solution. After mixing thoroughly, the solution was heated
for 10 min in water bath with constant temperature of 80 °C. The
sucrose concentration was obtained by the optical density (OD)
of the cooled solution at 500 nm. Cell concentration was measured
from OD value of culture broth at 660 nm. HA concentration was
measured by the carbazole method based on uronic acid determi-
nation (Bitter and Muir, 1962).
3. Theory and model development
3.1. Model development for batch cultivation of S. zooepidemicus
3.1.1. Microbial growth
Normally, substrate inhibited microbial growth can be
described by
8534 L. Liu et al. / Bioresource Technology 99 (2008) 8532–8536

lmax S dV
l¼ ð1Þ ¼F ð8Þ
K S þ S þ S2 =K I dt
dX F
where l is the specific growth rate (h1), lmax is the maximum spe- ¼ lX  X ð9Þ
dt V
cific growth rate (h1), S is the substrate concentration (g/L), KS is dP HA F
the substrate saturation constant (g/L) and KI is the substrate inhi- ¼ cHA X  P HA ð10Þ
dt V
bition constant (g/L).
dP LA F
The product inhibited microbial growth can be described by ¼ cLA X  P LA ð11Þ
dt V
S 1 dS F
l ¼ lmax ð2Þ ¼ kX þ ðPHA þ P LA Þ ð12Þ
K S þ S P=K IP dt V
where P is the product concentration (g/L) and KIP is the product where V is the broth volume (L), F is the feeding rate (mL/h), cHA is
inhibition constant (g/L). the specific HA synthesis rate (g HA/g cell/h), cLA is the specific lactic
In batch cultivation of S. zooepidemicus, cell growth is inhibited acid synthesis rate (lactic acid/g cell/h) and k is the specific sucrose
by both the substrate sucrose and the product lactic acid. There- consumption rate (g sucrose/g cell/h).
fore, the following model was proposed to describe the growth of
S. zooepidemicus in batch culture: 4. Results and discussion
lmax S 1
l¼ ð3Þ HA production by batch culture of S. zooepidemicus with an ini-
K S þ S þ S2 =K I 1 þ PLA =K ILA
tial sucrose concentration of 70 g/L was carried out. After 2 h of lag
where S is the sucrose concentration (g/L), KI is the sucrose inhibi- phase, cells entered the exponential growth phase until the end of
tion constant (g/L), PLA is the concentration of lactic acid (g/L) and 10 h and reached the maximum concentration of 13.2 g/L at 14 h.
KILA is the inhibition constant of lactic acid on cell growth (g/L). Sucrose was consumed for cell growth and metabolites synthesis,
and decreased to 8 g/L from an initial concentration of 70 g/L at
3.1.2. Product synthesis the end of fermentation. HA concentration reached the maximal
The main products in batch cultivation of S. zooepidemicus with value of 5 g/L at 16 h, and lactic acid was accumulated to 53 g/L
sucrose as carbon source are lactic acid and HA, and the synthesis at the end of batch culture. The maximum of specific cell growth
of HA and lactic acid are coupled with the cell growth. Further- rate was 1.21 h1. The average value of specific HA synthesis rate
more, lactic acid was inhibitive on the synthesis of itself and HA. was 0.024 (g HA/g cell/h) and that for lactic acid was 0.201 (g lactic
Therefore, the synthesis of HA and lactic acid can be described by acid/g cell/h). The yield of HA and lactic acid on sucrose were 0.08
Eqs. (4) and (5), respectively. (g HA/g sucrose) and 0.85 (g lactic acid/sucrose), respectively.
In the present study, a series of unstructured models of batch
dPHA aHA dX
¼ ð4Þ and fed-batch culture of S. zooepidemicus were developed based
dt 1 þ PLA =K HA dt
on the characteristics of HA fermentation. In these models, the
dPLA aLA dX inhibitions of sucrose and lactic acid on cell growth and products
¼ ð5Þ
dt 1 þ PLA =K LA dt (HA and lactic acid) synthesis were considered. With the obtained
model parameters (Table 1), the effectiveness of the developed
where PHA is the concentration of HA (g/L), PLA is the concentration unstructured models was verified and the developed models could
of lactic acid (g/L), aHA is the coupling constant of HA synthesis with describe the batch cultivation of S. zooepidemicus well (the average
cell growth, aLA is the coupling constant of lactic acid with cell value of R2 was 0.995).
growth, KHA is the inhibition constant of lactic acid on HA synthesis As indicated by the obtained inhibition constants of sucrose and
(g/L), KLA is the inhibition constant of lactic acid on the synthesis of lactic acid on cell growth, lactic acid had stronger inhibition on cell
itself (g/L) and t is the culture time (h). growth than sucrose. Meanwhile, it could be found that lactic acid
had stronger inhibition on cell growth than on HA synthesis. How-
3.1.3. Sucrose consumption ever, at the initial phase of batch culture, the inhibition of sucrose
Substrate consumption kinetics can be described by the follow- with high concentration on cell growth was predominant while the
ing model, which considered substrate conversion to cell mass and inhibition of cell growth by lactic acid of low concentration was al-
product as well as substrate consumption for maintenance: most negligible. Therefore, the cell growth at the initial phase of
batch culture could be described by Eq. (1). For this kind of sub-
dS 1 dX 1 dP HA 1 dP LA
 ¼ þ þ þ me X ð6Þ strate inhibited cell growth, as indicated by Eq. (7), there is an opti-
dt Y X dt Y HA dt Y LA dt
mal substrate concentration Sopt, at which the growth rate of the
where YX is the yield of cell on sucrose (g cell/g sucrose), YHA is the cells reached the maximum. For the batch culture of S. zooepidem-
yield of HA on sucrose (g HA/g sucrose), YLA is the yield of lactic acid icus with sucrose as carbon source, the value of Sopt is 1.0 g/L.
on sucrose (g lactic acid/g sucrose), me is the maintenance constant The influence of fed-batch culture with constant feeding rate
(h1) and X is the cell concentration (g/L). (22.5 mL/h) on HA production was studied (Fig. 1). The developed
The model parameters were obtained by Runge–Kutta algo- unstructured models could describe the fed-batch culture well (the
rithm with MATLAB 6.5.1 software. average R2 value was 0.993). The highest cell concentration in con-
For substrate inhibited growth described by Eq. (1), the specific stant feeding rate fed-batch culture was 16.3 g/L, which was higher
cell growth rate reaches the maximum value at S = Sopt: 23% than that in batch culture. Unexpectedly, HA production in
pffiffiffiffiffiffiffiffiffiffi fed-batch was only 4.0 g/L compared with 5.0 g/L in batch culture.
Sopt ¼ K S K I ð7Þ
The concentration of lactic acid was increased to 60 g/L compared
with 53 g/L in batch culture. The average specific HA synthesis rate
3.2. Model development for fed-batch cultivation of S. zooepidemicus in fed-batch culture was 0.014 (g HA/g cell/h) compared with 0.024
(g HA/g cell/h) in batch culture, while that for lactic acid in fed-
According to the substrate balance, the following models were batch culture was 0.208 (g lactic acid/g cell/h) compared with
proposed to describe the fed-batch cultivation of S. zooepidemicus: 0.201 (g lactic acid/g cell/h) in batch culture. The specific HA
 L. Liu et al. / Bioresource Technology 99 (2008) 8532–8536 8535

Table 1
Model parameters

Parameters lmax KS KI KILA aHA aLA KLA KHA YX YHA YLA me

Value 1.23 0.04 24.56 38.95 1.34 16.76 22.13 10.12 0.18 0.08 0.83 0.51
Units h1 g/L g/L g/L – – g/L g/L g/g g/g g/g h1

12 70 competition between HA synthesis and cell growth for the com-

Cell and lactic acid concentration (g/L)

mon precursors UDP-glucuronic acid and UDP-N-Acetyl Glucosa-
Sucrose and HA concentration (g/L)

60 mine. It should be noted that cell wall is necessary for cell

growth, while HA as a cell capsule is not a necessary component
50
8 for cell growth. Carbon source was utilized preferably for cell
40 growth and thus HA synthesis is inhibited, especially at low sub-
strate concentration in fed-batch culture. Therefore, a lower HA
30 productivity in fed-batch culture than batch culture was observed
4 in the present study. The competition between cell growth and HA
20
synthesis was also observed by other researchers (Fong Chong
10 et al., 2005). Therefore, the lower HA productivity in fed-batch cul-
ture resulted from the competition between cell growth and
0 0 HA synthesis, not the decreased HA synthesis ability of S.
0 4 8 12 16 20 zooepidemicus.
Culture time (h) Since there is a competition between cell growth and HA syn-
thesis, the alleviation of the competition may be an efficient strat-
Fig. 1. Time courses of HA production by S. zooepidemicus in fed-batch culture,
egy for the enhancement of HA production. As discussed above,
which was initiated as a batch culture with an initial sucrose concentration of 10 g/
L. The concentrated sucrose solution (600 g/L) was fed at a feeding rate of 22.5 mL/h
fed-batch culture was favorable for cell growth, and cells in fed-
during 2–20 h. Line: Model prediction; Symbols, experimental data (s: HA; :  batch culture can reach a concentration as high as that in batch
Sucrose; 4: cell; $: lactic acid). culture with a shorter time. As indicated by the parameters in Ta-
ble 1, lactic acid had higher inhibition on cell growth than HA syn-
thesis (the inhibition constant of lactic acid on cell growth KILA was
synthesis rate in fed-batch culture was 42% lower than that in four times higher than the inhibition constant of lactic acid on HA
batch culture, while the specific lactic acid synthesis rate of S. zoo- synthesis KHA). Sucrose also had high inhibition on cell growth.
epidemicus in fed-batch culture was the same with that in the Therefore, cell growth can be inhibited by lactic acid and sucrose
batch culture. Therefore, it seemed that fed-culture was more of high concentrations in batch culture and HA production can be
favorable for cell growth than batch culture, which was a more enhanced with the decreased competition of cell growth and HA
favorable way for HA synthesis. Then an important issue was synthesis. A two-step culture strategy, in which fed-batch culture
raised: which factor was responsible for the lower HA productivity was applied in the first phase for cell growth and batch culture
in fed-batch culture than batch culture? was carried out in the second phase for HA production, was pro-
UDP-Glucuronic acid and UDP-N-Acetyl Glucosamine are the posed in this study. As mentioned above, there was an optimal su-
two precursors for HA synthesis. Glucose-1-P and Glucose-6-P, crose concentration (1.0 g/L) for cell growth and thus the sucrose
which are the precursors of UDP-Glucuronic acid, also are the pre- concentration could be maintained at 1.0 g/L by a feedback control
cursors for lipopolysaccharide (cell wall) and teichoic acid, respec- strategy in the first phase to realize the fast cell growth. HA fer-
tively. Meanwhile, UDP-N-Acetyl Glucosamine is also the precursor mentation belonged to a kind of high viscosity cultivation and dis-
for peptidoglycan (cell wall) synthesis. Therefore, there existed a solved oxygen (DO) level decreased to nearly zero during

a 80 18 b 60 18
Cell and HA concentration (g/L)

15 50 15
Lactic acid concentration (g/L)

Sucrose concentration (g/L)

60
Feeding rate (mL/h)

12 40 12

40 9 30 9

6 20 6
20
3 10 3

0 0 0 0
0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
Culture time (h) Culrture time (h)

Fig. 2. Time courses of two-stage fed-batch and batch culture of S. zooepidemicus for HA production. Fed-batch culture was carried out in the first stage (0–8 h) and then the
sucrose concentration of culture broth was increased to 15 g/L to start the batch culture in the second stage (8–20 h). In the fed-batch culture process with the redox potential
level as the feedback control variable, the sucrose concentration of culture broth was maintained at 1.2 g/L by feeding concentrated sucrose solution (600 g/L). Fig. 2(a) s:
cell; : lactic acid; N: HA; Fig. 2(b) s: feeding rate; : sucrose.
8536 L. Liu et al. / Bioresource Technology 99 (2008) 8532–8536
exponential phase. The pH of the culture broth was maintained
constant at 7.0 during the whole process. Thus DO and pH could
not be used as a feedback variable to control sucrose concentration.
As an important control variable for fermentation process, redox
potential is attracting more and more attentions (Lin et al., 2005;
Sheu et al., 2003; Yu et al., 2007). In the present study, the redox
potential was used as a feedback control variable to maintain the
sucrose concentration at 1.0 g/L. The accuracy of the selected con-
trol strategy was further verified by the followed measurement of
sucrose concentration off line.
Fig. 2 shows the kinetics of two-stage culture of S. zooepidemicus
with the constant sucrose concentration at 1.0 ± 0.2 g/L in the first
phase of fed-batch culture. At 8 h, the cell concentration reached
13.5 g/L, which was the maximum cell concentration reached at
14 h in batch culture. The second phase of batch culture started
at 8 h with the increased sucrose concentration of 15 g/L until
the fermentation was over at 20 h. In the two-stage culture, 75 g/L
of total sucrose was used. When the batch culture with an initial
sucrose concentration of 75 g/L was carried out, the concentration
of HA and lactic acid were 5.1 g/L and 56 g/L, respectively, almost
the same with those in batch culture with an initial sucrose con-
centration of 70 g/L. With the proposed two-stage culture strategy,
HA production was increased to 6.6 g/L compared with 5.0 g/L in
the batch culture. The specific HA synthesis rate was 0.023
(g HA/g cell/h) and almost the same with that in batch culture.
The yield of HA on sucrose was increased to 0.092 (g HA/g sucrose)
in two-stage culture while that in batch culture was 0.071 (g HA/
g sucrose). Meanwhile, the sucrose utilization rate in the two-stage
culture was higher 14.2% than that in batch culture. Therefore, the
enhanced HA production in the two-stage culture resulted from
the increased sucrose transformation to HA. The significant
improvement of HA production proved the effectiveness of the
proposed model-based two-stage cultivation strategy. The culture
model developed in this study can be applied in high cell density
cultivation and provide an alternative strategy for the fermentation
processes, especially those where there exists a competition be-
tween cell growth and metabolites production for carbon source
and energy.
Acknowledgements
This project was financially supported by Program for Changji-
ang Scholars and Innovative Research Team in University
(No.IRT0532), the National Science Fund for Distinguished Young
Scholars of China (No.20625619) and 973 Project (2007CB714306).
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